Fermentation product A83543, a family of related factors produced by Saccharopolyspora spinosa, was recently discovered and was shown to exhibit excellent insecticidal activity. In European Patent Publication No. 0 375 316 fermentation product A83543 was shown to comprise individual components A83543A, A83543B, A83543C, A83543D, A83543E, A83543F, A83543G, A83543H, and A83543J. The general structure of the A83543 factors consists of a 5,6,5-tricyclic ring system fused to a 12-membered macrocyclic lactone, a neutral sugar, and an amino sugar. Kirst et al., Tetrahedron Letters, 32, 4839 (1991).
The U.S. patent application of Jon S. Mynderse et al. on "New A83543 Compounds and Processes for Production Thereof" (U.S. application Ser. No. 07/790,287) the same date as this application is hereby incorporated herein by reference. That application discloses factors A83543L, A83543M, and A83543N.
The following table identifies by structure these known A83543 components.
__________________________________________________________________________ ##STR1## Factor R.sup.3 R.sup.1 R R.sup.4 R.sup.5 R.sup.6 __________________________________________________________________________ A83543A H CH.sub.3 ##STR2## C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 A83543B H CH.sub.3 ##STR3## C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 A83543C H CH.sub.3 ##STR4## C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 A83543D CH.sub.3 CH.sub.3 ##STR5## C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 A83543E H CH.sub.3 ##STR6## CH.sub.3 CH.sub.3 CH.sub.3 A83543F H H ##STR7## C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 A83543G H CH.sub.3 ##STR8## C.sub.2 H.sub.5 CH.sub.3 CH.sub.3 A83543H H CH.sub.3 ##STR9## C.sub.2 H.sub.5 H CH.sub.3 A83543J H CH.sub.3 ##STR10## C.sub.2 H.sub.5 CH.sub.3 H A83543L CH.sub.3 CH.sub.3 ##STR11## C.sub.2 H.sub.5 CH.sub.3 H A83543M H CH.sub.3 ##STR12## C.sub.2 H.sub.5 CH.sub.3 H A83543N CH.sub.3 CH.sub.3 ##STR13## C.sub.2 H.sub.5 CH.sub.3 H __________________________________________________________________________
A83543 and each of the components are useful for the control of mites and insects, particularly Lepidoptera and Diptera species.
The term "A83543 component" means a compound selected from the group consisting of A83543A, A83543B, A83543C, A83543D, A83543E, A83543F, A83543G, A83543H, A83543J, A83543L, A83543M, and A83543N. If additional factors of the similar structure are subsequently discovered, it is contemplated that the isolation process provided by this invention will be applicable to them as well.
A83543 is produced by culturing an A83543-producing strain of Saccharopolyspora spinosa under submerged aerobic conditions by methods disclosed in the copending U.S. patent application of LaVerne D. Boeck et al., on "A83543A Compounds", U.S. application Ser. No. 07/773,754, filed Oct. 10, 1991 "A83543 Compounds," Ser. No; 07/429,441, filed Oct. 30, 1989, now abandoned and the above identified application of Jon S. Mynderse et al. on "New A83543 Compounds and Processes for Production Thereof" (U.S. application Ser. No. 07/790,287).
The term "A83543-producing strain of Saccharopolyspora spinosa" means a strain of Saccharopolyspora spinosa capable of producing recoverable amounts of at least one A83543 component. The following six A83543-producing Saccharopolyspora spinosa cultures, A83543.1, A83543.3, A83543.4, A83543.5, A83543.6, and A83543.7,have been deposited and made a part of the stock culture collection of the Midwest Area Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, from which they are available to the public under the following accession numbers:
______________________________________ NRRL No. Strain No. ______________________________________ 18395 A83543.1 18537 A83543.3 18538 A83543.4 18539 A83543.5 18719 A83543.6 18720 A83543.7 ______________________________________
The characteristics of these cultures are described in detail in the above identified U.S. patent applications of LaVerne D. Boeck et al. and Jon W. Mynderse et al. Each of the strains A83543.3, A83543.4, A83543.5, A83543.6, and A83543.7 was derived from A83543.1 by means described in those patent applications.
As is the case with other organisms, the characteristics of the A83543-producing strains are subject to variation. Thus, mutants of these strains may be obtained by physical and chemical methods known in the art. For example, other strains may be obtained by treatment with chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine. Natural and induced mutants derived directly or indirectly from A83543.1 which retain the characteristic of producing recoverable amounts of at least one A83543 component are applicable in the present invention.
The culture medium used to grow these Saccharopolypora spinosa cultures can be any one of a number of media. For economy in production, optimal yield, and ease of product isolation, certain culture media are preferred. For example, preferred carbon sources in large-scale fermentation are glucose and methyl oleate, although ribose, xylose, fructose, galactose, mannose, mannitol, maltose, soluble starch, potato dextrin, oils such as soybean oil and the like can also be used. Preferred nitrogen sources are cottonseed flour, peptonized milk and corn steep liquor, although fish meal, digested soybean meal, yeast extract, enzyme-hydrolyzed casein, beef extract, and the like can also be used. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding zinc, sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate and like ions. Essential trace elements necessary for the growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other substituents of the medium in amounts sufficient to meet the growth requirements of the organism.
Usually, if forming is a problem, small amounts (i.e., 0.2 ml/L) of an antifoam agent such as polypropylene glycol may be added to large-scale fermentation media. In the case of the A83543-producing cultures, however, conventional defoamers inhibit A83543 production. Foaming is controlled by including soybean oil or PLURONIC L-101 (BASF, Parsippany, N.J.) in the medium (1-3%). Additional oil is added if foaming develops.
The A83543 components are produced by the A83543-producing organisms when grown at temperatures between about 24.degree. and about 33.degree. C. Optimum temperatures for production appear to be about 28.degree.-30.degree. C.
As is customary in submerged aerobic culture processes, sterile air is blown into the vessel from the bottom while the medium is stirred with conventional turbine impellors. In general, the aeration rate and agitation rate should be sufficient to maintain the level of dissolved oxygen at or above 60% of air saturation, preferably above 65%, with an internal vessel pressure of about 0.34 atmospheres.
Production of the A83543 components can be followed during the fermentation by testing extracts of the broth. A preferred method for following the production is analysis of the broth extracts by high performance liquid chromatography (HPLC). A suitable system for analysis is described in Example 1.
The A83543 components produced during fermentation of the A83543-producing organism occurs in both the mycelial mass (the biomass) and the broth. A83543 components are lipophilic. Separation of the lipophilic A83543 components from the broth has proved to be a significant problem, because it requires a simultaneous separation of the components from the substantial quantity of oil used to control foaming during the fermentation.
Prior to this invention, A83532 components were isolated by the method described in U.S. patent application of Patrick J. Baker on "A83543 Recovery Process," Ser. No. 07/451,394, filed Dec. 15, 1989. Although the process described in that patent application is a significant improvement over prior processes, there remained a need to improve the efficiency of the recovery operation.